| Glioblastoma multiforme is the most prevalent type of primary brain tumor in adults, representing over 50% of all adult brain tumors. Despite recent advances in the development of chemotherapy, radiotherapy, and surgery, glioblastoma multiforme has an unfavorable prognosis and patient treatment remains one of the greatest challenges.Elemene, extracted from herb medicine Curcuma wenyujin, is a mixture of (3-, y-, and 8-elemene;β-elemene is the active component. Both in vitro and in vivo studies have demonstrated anti-tumor effects ofβ-elemene. It has been shown thatβ-elemene is an effective treatment for various types of cancer, including gastric, lung, ovarian cancers, and leukemia. Inhibition of cell proliferation and induction of apoptosis have been proposed as the underlying mechanism of the anti-tumor effects of P-elemene. We previously reported that treatment of glioblastoma cell lines with p-elemene led to cell-cycle arrest at G0/G1 phase and consequent inhibition of proliferation, and that the activation of p38MAPK was critical for the anti-proliferation effect ofβ-elemene. However, the mechanism by which P-elemene induces apoptosis of glioblastoma cells remains unclear. As the dynamic balance between growth factor-activated ERK and JNK-p38 pathways is important in determining whether a cell survives or undergoes apoptosis, the activation of p38MAPK and the deactivation of ERK are critical for the induction of apoptosis. In the present study, we investigated whether deactivation of the ERK pathway involvesβ-elemene-induced apoptosis of glioblastoma cells.Mitogen-activated protein kinase (MAPK) family members, including ERK(extracellular signal-regulated kinase, JNK(stress-activated c-JUN NH2-terminal protein kinase), and p38, mediate a multitude of cellular responses to extracellular stimuli (e.g. growth factors and hormones). The Ras/Raf/MEK/ERK cascade is an evolutionarily conserved pathway and plays a vital role in regulating many fundamental cellular processes, including proliferation, cell survival, differentiation, tumorigenesis, and development. Multiple members of the kinase families in this pathway can be activated or inactivated by protein phosphorylation. Ras activity is upregulated in the majority of cases of human glioblastoma multiforme. Furthermore, the level of phosphorylated ERK, a downstream effector of Ras, is also increased. Constitutive activation of the Ras/MAPK pathway is functionally important for glioblastoma cell proliferation.Extracellular signals, caused by the combination of ligands with their Receptor Tyrosine Kinases, are transducted to the interior of the cell via the switching of the Ras-family GTPases from the inactive GDP-bound state (Ras-GDP) to the active GTP-bound state (Ras-GTP). In its GTP-bound state, Ras activates the downstream signaling cascades—the Ras/Raf/MEK/ERK pathway. Raf (a serine/threonine kinase containing A-Raf, B-Raf, and C-Raf (also called Raf-1)) relays the extracellular signal from the receptor/Ras complex to the cytosolic kinase cascades by phosphorylating and activating MEK which, in turn, phosphorylates and activates ERK. Phosphorylated ERK then phosphorylates various cytoplasmic and nuclear proteins including members of the Bcl-2 family.The function of Ras-GTP is to phosphorylate and activate Raf kinase by guiding the accurate membrane location of Raf via the interaction with each other. Specifically, the amino acid regulatory domain of Raf-1(C-Raf) has a high affinity for Ras-GTP; once Raf-1 has translocated from the cytosol to the plasma membrane and combined with Ras-GTP, its amino acids at Tyr340 and Tyr341 are phosphorylated and activated by membrane-bound receptor tyrosine kinase. Activated Raf-1 (phosphorylated-Raf-1, p-Raf-1) alone is sufficient to phosphorylate and activate downstream MEK and ERK. In the process of Raf-1 activation described above,90 kDa heat-hock protein (Hsp90) plays an important role as chaperone. Raf-1 is one of the main client proteins for Hsp90, which may continuously form a stable "functional complex" with Raf-1 to keep its function stable as a special conformation. Thus, receptor tyrosine kinase may effectively activate Raf-1. In the absence of Hsp90, the amino acid regulatory domain of Raf-1 can not be exposed, Raf-1 thus loses its high affinity for Ras-GTP and fails to translocate from the cytosol to the plasma membrane and to bind Ras-GTP. In short, the association with Hsp90 is essential for both Raf-1 protein stability and its proper cellular localization.Hsp90 has become an important target for treatment of tumors. The benefit of Hsp90 inhibitors includes (1)their ability to inhibit many "mission critical" cancer pathways, (2) blockade of all "hallmark traits" of malignancy, and (3) the presentation of broad-spectrum antitumor effects.The Bcl-2 family proteins play key roles in the regulation of apoptosis. These proteins are either pro-apoptotic (e.g. Bax, Bad, and Bak) or anti-apoptotic (e.g. Bcl-2, Bcl-XL, and Mcl-1). An increase in the Bax:Bcl-2 ratio (an indicator of commitment to apoptosis) is known to promote mitochondrial release of cytochrome c into the cytosol which activates cysteine proteases, leading to apoptosis. Bcl-2 is one of many downstream signal molecules in the ERK1/2 pathway. ERK could serve as the responsible kinase for the phosphorylation and activation of Bcl-2.Objective:In this study, we examined the effects ofβ-elemene on the activation of Raf/MEK/ERK pathway, the expression of Bcl-2, and the formation of Hsp90/Raf-1 molecular complex. We investigated the mechanism ofβ-elemene -induced apoptosis of rat C6 glioma cells and human U87 glioma cells in vitro and in vivo.Methods:Using the methods of cell counting, flow cytometry, Co-immunoprecipitation assay, and Western blotting analysis, we examined the effect of elemene with different concentrations on proliferation and apoptosis of C6 and U87 cells, the expression of protein Raf-1, ERK, Bcl-2, and Bax in C6 and U87 cells, the recruitment level of Hsp90 in the Hsp90/Raf-1 complex; meanwhile, we also investigated the effect of elemene on tumor growth in U87 cell-transplanted tumors in nude mice.Results:We found that:(1)β-elemene dose-and time-dependently suppressed proliferation of C6 glioma cells in vitro; (2)β-elemene time-dependently induced the apoptosis of C6 and U87 cells; (3)β-elemene decreased the recruitment of Hsp90 in the Hsp90/Raf-1 molecular comple and disrupted the formation of the Hsp90/Raf-1 molecular complex; (4)β-elemene downregulated the expression of phosphorylated Raf-1 and ERK in C6 and U87 cells(dose dependently); (5)β-elemene downregulated the expression of Bcl-2(dose dependently), did not alter the level of Bax, increased the ratio of Bax/Bcl-2; (6) Intraperitoneal injection ofβ-elemene also suppressed the tumor growth in U87 cell-transplanted tumors in nude mice.Conclusions:(1)β-elemene dose-and time-dependently inhibited the proliferatin of C6 glioma cells in vitro; (2)In vitroβ-elemene time-dependently induced the apoptosis of C6 and U87 cells; (3)β-elemene disrupted the Hsp90/Raf-1 molecular complex; the prevention of Hsp90/Raf-1 complex formation damaged Raf-1 protein stability and prevented activation of Raf-1. In turn, deactivation of Raf-1 inhibited the Raf/MEK/ERK pathway, leading to apoptosis of glioblastoma cells; (4)β-elemene inhibited the tumor growth in U87 cell-transplanted tumors in nude mice. These results suggest that the Hsp90/Raf-1 complex could be a promising molecular target for new drug development for the treatment of glioblastoma. |
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